The Automated Cytometry and Cell Sorter Laboratory/Confocal Microscopy and Image Analysis Facility provides cellular analysis to investigators with peer-reviewed grants at M.D. Anderson Cancer Center. The facility develops and provides cutting-edge techniques in single-cell analysis. Cell phenotyping, proliferation, and apoptosis assays have been established and modified as needed for multi-parameter analysis. Immunophenotypic analysis was combined with assays of intracellular proteins related to apoptosis (bcl-2, BAG-1, Bcl-Xl, p53, Rb, and fas), proliferation and membrane lipid asymmetry. Quantitation of cellular antigens allow determination of antibody binding capacity per cell. Very rare events and progenitor cell subpopulations have been detected and isolated by three-laser excitation/eight-parameter fluorescence-activated cell sorting (FACS) for subsequent analysis by molecular cytogenetics and other molecular techniques. Acquisition of a high-speed cell sorter and a laser-scanning microscope will provide state-of-the-art isolation and analysis. Laser confocal microscopy has been used extensively, and so the acquisition of a second instrument is necessary. By using a charge coupled-device (CCD)- based image analysis system, a method for image capture in perfect registration was developed and has been used extensively for molecular cytogenetics fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). FISH has also been combined with the apoptosis assay to detect apoptosis in normal and leukemic cells. The number, phenotype, and proliferation of minimal residual disease cells with abnormalities amenable to FISH analysis can be determined at levels of as few as one malignant cell in 30,000 normal cells. Methods to detect transgene expression in cells have been established using beta- galactosidase (beta-gal), nerve growth factor receptor (NGF-R), and green fluorescent protein. The Facility has served 26 investigators with peer- reviewed grants who used the laser confocal microscope for 1,797 and the FACS facility for 1,970 hours last year, a 33% increase over the last 2 years. The Core has continuously developed new methodology to suit the evolving needs of its users.